TaqMan™-PCR for the detection of pathogenic E. coli strains

ABSTRACT

The present invention relates to a method for the detection of pathogenic  E. coli  in a sample comprising PCR amplification of DNA isolated from said sample using oligonucleotide primers specific for pathogenic  E. coli.

The present invention relates to a rapid, high performance assay for thedetection of pathogenic E. coli which is based on TaqMan™ PCR technique,and to specific optimised oligonucleotide primers and labelledoligonucleotide probes useful in the assay.

BACKGROUND OF THE INVENTION

Enterohemorrhagic, shiga-like toxin (sit) producing Escherchia coli(EHEC) have recently been recognized as an important human and animalpathogen (1-7). EHEC has been responsible for several food-borneoutbreaks (8). The most notable were a multistate outbreak associatedwith a fast food chain in the western states of the USA with more than600 individuals affected and 3 deaths in Washington (9), and an epedemicoccurence in Japan with more than 6000 patients and approx. 8 fatalcases (10). Infection with EHEC causes diarrhea, hemorrhagic colitis,thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome (HUS)that is characterised by acute renal failure, thrombocytopenia, andmicroangiopathic hemolytic anemia. HUS ultimately can result in a fataloutcome in affected children and immunocompromised individuals(3,11-17). Recently, in the South-Eastern parts of Germany (Bavaria) anincrease of EHEC cases was reported during October 1995 and July 1996with at least 45 severe infections leading to HUS accompanied by 7deaths (18). Estimating that approx. 1 out of 15 EHEC infections resultsin HUS approx. 600-700 affected individuals might be assumed.

In most outbreaks reported, consumption of contaminated ground beef hasbeen the source of infection (5,8,19-22), whereas in Japan raddishsprouts are suspected (10). EHEC has been isolated from cow milk(6,19,23), water (19), chicken, pork, and apple cider (19,24,25), butalso human horizontal smear infections have been reported (15). Cattleappear likely to be the reservoir (22,26). Cross contamination, improperhandling, and inadequate cooking all contribute to food-borne infectionscaused by EHEC. EHEC produce Shiga-like toxins (slt), also known asverotoxins or cytotoxins (12,27). A large proportion of EHEC have beenfound to belong to the serogroup O157:H7, but notably, also a variety ofEHEC belonging to other serogroups (O22, O26, O55, O111, O114, O145)have been reported especially in Europe (12,15,28-32).

Besides EHEC, certain other strains of E. coli can cause enteritis orgastroenteritis and are grouped in enterotoxigenic strains (ETEC)(33-36), enteropathogenic strains (EPEC) (37), enteroinvasive strains(EIEC) (38,39), and enteroaggregative strains (EaggEC) (40,41). Thesestrains are important pathogens and also pose severe public healthproblems. The diagnosis of these pathogens is vastly neglegted due tothe lack of specific and sensitive routine test methods. ETEC synthesizeheat labile and/or heat stable enterotoxins that can cause a secretorydiarrhea (“traveller's diarrhea”) resembling that of Vibrio cholerae(36,42,43). Surface attachment of the ETEC organisms to the intestinalepithelial cell is a prerequisite to toxin production. Toxin productionis plasmid mediated and most commonly involves E. coli serogroups O6,O15, O124, O136, O143, O145, and 0147 (32). EPEC cause diarrhealsymptoms primarily in infants (32). Although the pathogenesis isunclear, the epithelial degradation of the gut, and the inflammatoryresponse that are observed in tissue sections may be a consequence dueto the adhesive properties of the bacterium. Specific attachment factorsof EPEC are plasmid encoded (EAF=EPEC adherence factor) (37,44). EHECoften contain an adherence factor closely related to EAF that is knownas ene (EHEC attaching and effacing gene) (45,46). EPEC most oftenbelong to serogroups O6, O8, O25, O111, O119, and O142 (32).

EIEC strains are capable of penetrating and invading the intestinalepithelial cells and produce an inflammatory diarrhea similar to thatcaused by Shigella bacteria (38,47,48). Fecal smears contain blood,mucus and segmented neutrophils. EIEC contain virulence plasmids codingfor additional pathogenic factors (48). Serogroups O28, O112, O115,O124, O136, O143, O145, and O147 are most commonly found on EIEC (32).

EaggEC are associated with persistent diarrhea in children and withtraveller's diarrhea. EaggEC are characterized by their adherencecapacity that leads to aggregation of Hep-2 cells. This effect isassociated with the presence of a virulence plasmid (pCVD432). EaggECare supected to also produce a heat stable enterotoxin (EAST1) (49-53).They can belong to serogroups O44 and O126 (32).

Conventional detection methods for EHEC encompass enrichment andisolation with selective and/or indicator media such as E. coli broth,lauryl sulfate tryptose 4-methylumbelliferyl-b-acid broth, eosinmethylene blue agar, McConkey sorbitol agar, and enterohemolysin agar(28,32,54-59). All of these assays, unfortunately, are indirect and lackthe ability to identify EHEC or the other pathogenic E. coli strainsspecifically. Several methods for biochemical identification andimmunological detection of EHEC have been put forward (54,60-63),however, it is well recognized that pathogenic E. coli strains neitherposess nor lack unique fermentation pathways (58,64). Serotyping is notconclusive since no absolute correlation between serotype and pathogenicE. coli group can be established (12,27,32,58,65). DNA hybridizationtechniques have been established for experimental research but are notapplicable for large scale routine diagnostic procedures (66,67). DNAamplification based assays, using PCR have been reported (68-72).Limitations to these methods include cumbersome post-PCR detectionmethods (agarose gel electrophoresis, Biotin/Avidin based ELISAdetection systems).

To overcome these problems, a PCR assay which allows the specificdetermination of virulence factors characteristic for EHEC, ETEC, EPEC,EIEC, and EaggEC that is based on a fluorigenic detection method of PCRamplification has been developed.

This assay exploits the 5′→3′ exonuclease activity of Taq-DNA polymerase(73) to cleave an internal oligonucleotide probe that is covalentlyconjugated with a fluorescent reporter dye (e.g. 6-carboxy-fluorescein[FAM]; λ_(em)=518 nm) and a fluorescent quencher dye(6-carboxytetram-ethyl-rhodamine [TAMRA]; λ_(em)=582 nm) at the 5′ and3′ end, respectively (74,75). Fluorescence from FAM is efficientlyquenched by TAMRA on the same, intact probe molecule (76). In the casethat cognate PCR amplification occurs, Taq polymerase extends from thespecific PCR primer and cleaves the internal, fluorigenicoligonucleotide probe annealed to the template strand. Thus, thereporter dye and the quencher dye get spatially separated. As aconsequence of oligonucleotide hydrolysis and physical separation of thereporter and the quencher dyes, a measurable increase in fluoresecenceintensity at 518 nm can be observed. PCR cycling leads to exponentialamplification of the PCR product and consequently of fluorescenceintensity.

TAQMAN™-PCR is performed in optical tubes that allow measurements offluorescence signals without opening the PCR tubes. This dramaticallyminimizes post-PCR processing time and almost completely eliminatescross-PCR contamination problems. Employing this approach, simultaneoustesting of biological materials for the presence of virulence genes ofLcoh strains and other enterobacteria, harboring virulence genes can besemiautomated and performed within 18 h.

According to the present invention Real Time PCR (e.g., TAQMAN™ PCR) forthe detection of pathogenic E. coli is provided, enabling for the firsttime the specific, rapid and high throughput routine detection of EHEC,ETEC, EPEC, EIEC, and EaggEC and related enterobacteria that harborthese virulence genes in routine bacteriological laboratories.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a rapid, highperformance assay for the detection and identification of pathogenic E.coli in biological samples.

It is a further object of the present invention to provide specific,optimised primers and labelled oligonucleotide probes useful for theamplification of sequences encoding virulence factors/toxinscharacteristic for pathogenic E. coli

SUMMARY OF THE INVENTION

The invention then, inter alia, comprises the following alone or incombination:

A method for the detection of pathogenic E. coli in a sample comprisingPCR amplification of DNA isolated from said sample using a set ofoligonucleotide primers specific for virulence factors/toxins ofpathogenic E. coli selected from

primers that hybridise to a gene encoding heat labile toxin, or heatstabile toxin for the amplification of a DNA sequence characteristic forenterotoxigenic E. coli;

primers that hybridise to a gene encoding heat stabile toxin for theamplification of a DNA sequence characteristic for enteroaggregative E.coli;

primers that hybridise to the pCVD432 plasmid for the amplification of aDNA sequence characteristic for enteroaggregative E. coli;

primers that hybridise to the inv-plasmid for the amplification of a DNAsequence contained in enteroinvasive E. coli;

primers that hybridise to the EAF plasmid, or the eae gene for theamplification of a DNA sequence characteristic for enteropathogenic E.coli; and/ or

primers that hybridise to the genes encoding shiga-like toxin sltI orsltII for the amplification of a DNA sequence characteristic forenterohemorrhagic E. coli, followed by detection and identification ofthe amplified product using conventional methods;

the method as above wherein

the set of primers that hybridise to the gene encoding heat labile toxincharacteristic for enterotoxigenic E. coli is

LT-1: ^(5′)GCG TTA CTA TCC TCT CTA TGT G^(3′) (SEQ ID NO:1) and

LT-2: ^(5′)AGT TTT CCA TAC TGA TTG CCG C^(3′) (SEQ ID NO:2);

the set of primers that hybridise to the gene encoding heat stabiletoxin characteristic for enterotoxigenic E. coli is

ST-1: ^(5′)TCC CTC AGG ATG CTA AAC CAG^(3′) (SEQ ID NO:3) and

ST-2a: ^(5′)TCG ATT TAT TCA ACA AAG CAA C^(3′) (SEQ ID NO:4);

the set of primers that hybridise for the gene encoding heat stabiletoxin characteristic for enteroaggregative E. coli is

EASTI-1: ^(5′)AAC TGC TGG GTA TGT GGC TGG^(3′) (SEQ ID NO:5) and

EASTI-2: ^(5′)TGC TGA CCT GCC TCT TCC ATC^(3′) (SEQ ID NO:6);

the set of primers which hybridise to the pCVD432 plasmid is

EA-1: ^(5′)CTG GCG AAA GAC TGT ATC ATT G^(3′) (SEQ ID NO:7) and

EA-2: ^(5′)TAA TGT ATA GAA ATC CGC TGT T^(3′) (SEQ ID NO:8);

the set of primers which hybridise to the inv-plasmid is

EI-1: ^(5′)TTT CTG GAT GGT ATG GTG AGG^(3′) (SEQ ID NO:9) and

EI-2: ^(5′)CTT GAA CAT AAG GAA ATA AAC^(3′) (SEQ ID NO:10);

the set of primers which hybridise to the EAF plasmid is

EP-1: ^(5′)CAG GGT AAA AGA AAG ATG ATA AG^(3′) (SEQ ID NO:11) and

EP-2: ^(5′)AAT ATG GGG ACC ATG TAT TAT C^(3′) (SEQ ID NO:12)

the set of primers which hybridise to the eae gene is

EPeh-1: ^(5′)CCC GGA CCC GGC ACA AGC ATA AG^(3′) (SEQ ID NO:13) and

EPeh-2: ⁵′AGT CTC GCC AGT ATT CGC CAC C^(3′) (SEQ ID NO:14);

the primers which hybridises to the gene encoding shiga-like toxin SltIis

SltI-1: ^(5′)ATG AAA AAA ACA TTA TTA ATA GC^(3′) (SEQ ID NO:15) and

SltI-2: ^(5′)TCA CYG AGC TAT TCT GAG TCA AGC^(3′) (SEQ ID NO:16); and

the primers which hybridises to the gene encoding shiga-like toxin SltIIis

SltII-1: ^(5′)ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G^(3′) (SEQ IDNO:17) and

SltII-2: ^(5′)TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC^(3′) (SEQ IDNO:18)

wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T; themethod as above wherein a polymerase having additional 5′-3′ exonucleaseactivity is used for the amplification of DNA, and an oligonucleotideprobe labelled at the most 5′ base with a fluorescent dye and at themost 3′ base with a fluorescent quencher dye which hybridises within thetarget DNA is included in the amplification process; said labelledoligonucleotide probe being susceptible to 5′-3′ exonuclease degradationby said polymerase to produce fragments that can be detected byfluorogenic detection methods;

the method as above wherein the labelled oligonucleotide probe for thedetection of heat labile toxin characteristic for enterotoxigenic E.coli is

^(5′)AGC TCC CCA GTC TAT TAC AGA ACT ATG^(3′) (SEQ ID NO:19);

the labelled oligonucleotide probe for the detection of heat stabiletoxin characteristic for enterotoxigenic E. coli is

^(5′)ACA TAC GTT ACA GAC ATA ATC AGA ATC AG^(3′) (SEQ ID NO:20);

the labelled oligonucleotide probe for the detection of heat stabiletoxin characteristic for enteroaggregative E. coli is

^(5′)ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC^(3′) (SEQ ID NO:21);

the labelled oligonucleotide probe for the detection of pCVD432 plasmidis

^(5′)CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG^(3′) (SEQ ID NO:22);

the labelled oligonucleotide probe for the detection of the inv-plasmidis;

^(5′)CAA AAA CAG AAG AAC CTA TGT CTA CCT^(3′) (SEQ ID NO:23)

the labelled oligonucleotide probe for the detection of the EAF-plasmidis;

^(5′)CTT GGA GTG ATC GAA CGG GAT CCA AAT^(3′) (SEQ ID NO:24);

the labelled oligonucleotide probe for the detection of the eae gene is

^(5′)TAA ACG GGT ATT ATC AAC AGA AAA ATC C^(3′) (SEQ ID NO:25);

the labelled oligonucleotide probe for the detection of shiga-like toxinSltI gene is

^(5′)TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA^(3′) (SEQ ID NO:26); and

the labelled oligonucleotide probe for the detection of shiga-like toxinSltII gene is

^(5′)CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT^(3′) (SEQ ID NO:27);

the method as above wherein the fluoroscent reporter dye is6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein, orhexachloro-6-carboxy-fluoroscein, and the fluorescent quencher dye is6-carboxytetramethyl-rhodamine;

the method as above wherein the PCR amplification process consists of 35PCR cycles at a MgCl₂ concentration of 5.2 mmol, an annealingtemperature of 55° C. and an extension temperature of 65° C.;

a set of primers useful for PCR amplification of DNA specific forvirulence factors/toxins of pathogenic E. coli selected from:

a set of primers that hybridise to a gene encoding heat labile toxin, orheat stabile toxin of enterotoxigenic E. coli;

a set of primers that hybridise to a gene encoding heat stabile toxin ofenteroaggregative E. coli;

a set of primers that hybridise to the pCVD432 plasmid ofenteroaggregative E. coli;

a set of primers that hybridise to the inv-plasmid of enteroinvasive E.coli;

a set of primers that hybridise to the EAF plasmid, or the eae gene ofenteropathogenic E. coli; and

a set of primers that hybridise to the gene encoding shiga-like toxinsltI or sltII of enterohemorrhagic E. coli;

the set of primers as above wherein

the set of primers which hybridise to the gene encoding heat labiletoxin of enterotoxigenic E. coli is

LT-1: ^(5′)GCG TTA CTA TCC TCT CTA TGT G^(3′) (SEQ ID NO:1) and LT-2:⁵′AGT TTT CCA TAC TGA TTG CCG C^(3′) (SEQ ID NO:2);

the set of primers which hybridise to the gene encoding heat stabiletoxin of enterotoxigenic E. coli is

ST-1: ^(5′)TCC CTC AGG ATG CTA AAC CAG^(3′) (SEQ ID NO:3) and

ST-2a: ^(5′)TCG ATT TAT TCA ACA AAG CAA C^(3′) (SEQ ID NO:4);

the set of primers which hybridise to the gene encoding heat stabiletoxin of enteroaggregative E. coli is

EASTI-1: ^(5′)AAC TGC TGG GTA TGT GGC TGG^(3′) (SEQ ID NO:5) and

EASTI-2: ^(5′)TGC TGA CCT GCC TCT TCC ATG^(3′) (SEQ ID NO:6);

the set of primers which hybridise to the pCVD432 plasmid is

EA-1: ^(5′)CTG GCG AAA GAC TGT ATC ATT G^(3′) (SEQ ID NO:7) and

EA-2: ^(5′)TAA TGT ATA GAA ATC CGC TGT T^(3′) (SEQ ID NO:8);

the set of primers which hybridise to the inv-plasmid is

EI-1: ^(5′)TTT CTG GAT GGT ATG GTG AGG^(3′) (SEQ ID NO:9) and

EI-2: ^(5′)CTT GAA CAT AAG GAA ATA AAC^(3′) (SEQ ID NO:10);

the set of primers which hybridise to the EAF plasmid is

EP-1: ^(5′)CAG GGT AAA AGA AAG ATG ATA AG^(3′) (SEQ ID NO:11) and

EP-2: ^(5′)AAT ATG GGG ACC ATG TAT TAT C^(3′) (SEQ ID NO:12);

the set of primers which hybridise to the eae gene is

EPeh-1: ^(5′)CCC GGA CCC GGC ACA AGC ATA AG^(3′) (SEQ ID NO:13) and

EPeh-2: ^(5′)AGT CTC GCC AGT ATT CGC CAC C^(3′) (SEQ ID NO:14);

the set of primers which hybridise to the shiga-like toxin sltI gene is

SltI-1: ^(5′)ATG AAA AAA ACA TTA TTA ATA GC^(3′) (SEQ ID NO:15) and

SltI-2: ^(5′)TCA CYG AGC TAT TCT GAG TCA AGC^(3′) (SEQ ID NO:16);

and

the set of primers which hybridise to the shiga-like toxin sltII is

SltI-1: ^(5′)ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G^(3′) (SEQ IDNO:17) and

SltII-2: ^(5′)TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC^(3′) (SEQ IDNO:18)

wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T;

the set of primers as above which in addition to the primers foramplification of target DNA comprise a labelled oligonucleotide probewhich is labelled with a fluoroscent reporter dye, such as6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein,hexachloro-6-carboxy-fluoroscein, at the most 5′ base and a fluoroscentquencher dye, such as 6-carboxytetramethyl-rhodamine, at the most 3′base, and have a nucleotide sequence selected from

^(5′)AGC TCC CCA GTC TAT TAC AGA ACT ATG^(3′) (SEQ ID NO:19)

which hybridises to a gene encoding heat labile toxin of enterotoxigenicE. coli;

^(5′)ACA TAC GTT ACA GAC ATA ATC AGA ATC AG^(3′) (SEQ ID NO:20)

which hybridises to a gene encoding heat stabile toxin ofenterotoxigenic E. coli;

^(5′)ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC^(3′) (SEQ ID NO:21)

which hybridises to a gene encoding heat stabile toxin ofenteroaggregative E. coli;

^(5′)CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG^(3′) (SEQ ID NO:22)

which hybridises to the pCVD432 plasmid;

^(5′)CAA AAA CAG AAG AAC CTA TGT CTA CCT^(3′) (SEQ ID NO:23)

which hybridises to the inv-plasmid;

^(5′)CTT GGA GTG ATC GAA CGG GAT CCA AAT^(3′) (SEQ ID NO:24)

which hybridises to the EAF plasmid;

^(5′)TAA ACG GGT ATT ATC AAC AGA AAA ATC C^(3′) (SEQ ID NO:25)

which hybridises to the eae gene;

^(5′)TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA^(3′) (SEQ ID NO:26)

which hybridises to the shiga-like toxin SltI gene; and

^(5′)CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT^(3′) (SEQ ID NO:27)

which hybridises to the shiga-like toxin SltII gene;

the use of the method as above for diagnosing an E. coli infection of aliving animal body, including a human, or for the detection of E. colicontamination of consumables, such as meat, milk and vegetables.

The Invention

Conventional methods used to detect PCR amplification are laboursome,employ potentially carcinogenic substances (ethidium bromide gelelectrophoresis), and are not suited as a routine assay method in themicrobiological routine laboratory (68-72). This poses a seriousproblem, especially when potential pathogenic bacteria cannot bedifferentiated from facultative pathogenic or apathogenic ones due tocharacteristic biochemical, serological and/or morphological criteria.Thus, specific nucleic acid-based diagnostic methods that directlydetect virulence factors or toxins harbored by these species aremandatory. This is in principal the case for the diagnosis of pathogenicE. coli bacteria. Biochemical properties of EHEC, EPEC, EIEC, ETEC, andEaggEC are not unique and cannot be used for setting them apart fromother E. coli strains (54,60-62). Furthermore, virulence plasmids of E.coli can be found in other enterobacteria as well (38,48,83,88,89).Because of the diverse serological makeup, identification of pathogenicE. coli by serotyping is also not an accurate means of identification(12,15,28-32). Classical colony hybridization assays with probesspecific for characteristic virulence factor and/or toxin genes arelaborous and timeconsuming (66,67). Classical PCR methods requirevarious post-PCR steps in order to verify whether specific amplificationof a target gene has occured (68J2). The TAQMAN™-PCR detection system(74,75,90) enables the rapid, specific, sensitive, and high-throughputdiagnosis for differentiation of pathogenic Lcoli strains from otherstrains of E. coli. The assay has the ability to quantify the initialtarget sequence. Since PCR-reaction tubes have not to be opened afterPCR cycling, the potential danger of cross-PCR contamination is almostnegligible. The scanning time of 96 samples is approximately 8 min, andcalculation of test results can be automated with a commerciallyavailable spread sheet program. Thus, overall post-PCR processing timeis cut to a minimum.

The TAQMAN™-System relies on standard PCR technique with the addition ofa specific internal fluorogenic oligonucleotide probe. The combinationof conventional PCR with the Taq polymerase-dependent degradation of aninternally hybridized ohgonucleotide probe confers also specificity tothis detection method, since it is highly unlikely that unspecific PCRamplification will yield positive fluorescence signals. Some rules forchosing the fluorigenic probes have to be obeyed (74,75). Criticial arethe lenght of the probe, the location of reporter and quencher dyes andthe absence of a guanosine at the 5′-end (74). Also, the distance of theprobe from one of the specific PCR primers is important. This is due tothe fact that the probe has to stay annealed to the template strand inorder to be cleaved by Taq polymerase. Since annealing depends, at leastpartially, on the Tm of the probe, probes should be designed to have ahigher Tm as the primers. According to the present invention this wassolved (except for sltII) by designing probes that were 3 to 6 bp longerthan the specific primers. PCR amplification includes extension of thetarget sequence after annealing of the primers and the Tm of theextended primers increases. For the fluorogenic oligonucleotide probe,where the 3′- end is capped in order to avoid elongation, the Tm remainsconstant, making it more likely that the probe dissociates beforedegradation by Taq polymerase. Oligonucleotide probe degradation can beoptimized by spatial proximity of the fluorogenic probe and the primer.By moving the probe for SltI from 121 bp to 9 bp close to the primer, asignificant improvement in ARQ values could be obtained. A secondstrategy of optimization of TAQMAN™-PCR is to perform PCR elongation at65° C., where it is also less likely that the probe dissociates from thetemplate strand before Taq polymerase reaches and hydrolizes it. Valuesfor ΔRQ can thus again be increased about 1.2 to 1.5 fold. The increaseof ΔRQ values might be due to the ratio of annealed oligonucleotideprobe reached by Taq polymerase or to an increased processivity of Taqpolymerase.

The concentration of fluorogenic probes influences the accuracy ofTAQMAN™-results. When the probe concentrations were >50 pmol/PCRreaction only a relatively small fraction was hydrolysed by Taqpolymerase. The ratio of undegraded probe to degraded probe remains highand the fluorescence emmission of the unquenched reporter dye does notsignificantly increase in relation to the fluorescence intensity of thereporter dye sill close to the quencher. Thus, at high probeconcentrations, ARQ values are lower than with intermediate probeconcentrations (10 20 pmol). When the probe concentration is too low,ΔRQ values are increased, however, variability of PCR results isincreased, since probably small errors in pipeting or minimaldifferences between PCR reactions become critical. Optimal probeconcentration that yielded smallest variabilties and highest RQ valueswere found at a probe concentration of 20 pmol.

Since TAQMAN™-PCR uses an internal oligonucleotide probe for detectionof template amplification, specific primers and probes can be amplydesigned. The design of primer and probe sequences is especiallyimportant, when nucleotide sequence variants of a given gene exist. Thisis the case for sltI and sltII. For sltI, all published sequences werealigned and primers and probes were designed to bind to conservedregions of all three variants. For sltII, only one region of thepublished genes was conserved, thus this region was chosen for thefluorogenic oligonucleotide probe. The primers for amplification ofsltII were designed to contain all possible nucleotide sequences at theambiguous positions of the published sltll variants (degenerate primerapproach) (79-83). By employing degenerate primers, it is possible todetect all published variants in one single PCR reaction.

The isolation method for template DNA affects the performance of thePCR. Two methods, that are suited as rapid purification steps forroutine applications, namely boiling prep or spin prep were compared.Boiling preps may still contain some bacterial components that canaffect PCR reactions, however, it is extremely fast. The spin prepmethod involves isolation steps that serve to purify DNA frompotentially negatively influencing materials. ΔRQ values and sensitivityof TAQMAN™-PCR for virulence genes from enterobacteria was not foundsignificantly increased as compared to boiling preps when template DNAwas prepared by spin prep method. The overall sensitivity of TAQMAN™-PCRfor all primer/probe combinations was comparable to visual scoring ofPCR products by detection with ethidium bromide stained agarose gelelectrophoresis. Under optimized. conditions, as few as 10³ cfusltI+EHEC could be detected among 10⁷ non-pathogenic E. coli per PCRreaction.

The use of immunomagnetic detection methods for E. coli O157 (54,91) hasbeen put forward as a means to improve sensitivity of EHEC diagnosticsby enrichment of this serogroup since the first sit producing strainswere found to be O157:H7 positive (1,2). However, it is obvious thatEHEC that are O157 antigen negative will be missed by this method. Itbecame clear during serotyping studies of recent EHEC isolates that thenumber of O157+EHEC now is small as compared to non-O157 EHEC(12,15,28,29,31). In a recent study, conducted in Southern Germany only2 of 13 isolates were O157 positive (92). Immunomagnetic detectionmethods for other O serotypes are currently not available. Also, otherenterobacteria such as Citrobacter sp. (83) and Enterobacter sp. (89)that can harbor shiga like toxins would be missed in the case of biasedenrichment procedures previous to analysis of virulence genes. Thus,TaqMan™-based PCR that is designed for detection of virulence genes inall enterobacteria appears to be superior.

The infectious agents of a large proportion of diarrheal diseases is notknown. Routine screening for bacterial pathogens in the gastrointestinaltract encompasses Salmonella sp, Shigella sp, S. aureus, Campylobactersp., Vibrio sp., Yersinia sp., and C. difficile (32). It is wellrecognized that pathogenic E. coli such as ETEC, EHEC, EIEC, and EaggECare important pathogens of the lower gastrointestinal tract andtherefore might significantly contribute to the number of diarrhealinfections (32). However, no routine bacteriological diagnosticprocedures for these bacteria are performed, and, moreover, in mostcases these pathogenic E. coli are misdiagnosed under the category ofnon-pathogenic “commensal flora”. In order to address this problem a setof specific primers and fluorogenic probes were developed and optimizedfor TAQMAN™-based detection of virulence factors harbored by thesebacteria (Tables 2 and 3). Arranging patient samples, positive andno-template controls of all 8 tested virulence genes in a standard 96well microtiter format, a turnaround time from preparation of sample DNAto fluorescence measurement of under 5 hours can be achieved. Thus, theTAQMAN™-based assay for pathogenic E. coli provides an ultrarapid meansof diagnosis of these bacteria. While being accurate, sensitive andspecific, this assay requires minimal post PCR processing time comparedto conventional methods. When TAQMAN™-PCR is performed in optical tubesalso the danger of cross-contamination of PCR reactions with amplifiedproducts is reduced to a minimum. Detection of virulence plasmidsharbored by pathogenic enterobacteria might prove the potential of thesebacteria to cause disease in the host. It is not clear whetherenterobacteria that contain toxin genes or attachment factors do alsoalways express them outside the host. This might be an explanation whyELISA tests for shiga like toxins might be negative in a number of HUScases where sltI and/or sltII containing EHECs can be detected bynucleic acid based methods.

The TAQMAN™-assay according to the invention for detection of pathogenicE. coli was then tested in a routine diagnostic setting for theexamination of stool samples obtained from children with diarrhea withina defined geographic area (Southern Bavaria) during a 7 month period.Results obtained by TAQMAN™-PCR were compared to the standard detectionmethod for PCR products (electrophoresis of ethidium stained agarosegels). 100 stool samples were analysed (Table 4). 22% of samples werefound to test positive for one or more virulence factors. There were 2cases. of EHEC, 5 ETEC, 8 EaggEC, 1 EIEC, and 16 EPEC. This means that ⅕of children with diarrhea probably suffered from diarrhea caused bypathogenic E. coli. These numbers are far higher than these for allother groups of routinely screened bacterial gastrointestinal tractpathogens. Only 2 cases of salmonella and no campylobacter were observedwithin this group.

Interestingly, the two children diagnosed with EHEC were severely sick,one suffered from hemorrhagic colitis, the other developed HUS and hadto be treated in a critical care unit.

Collectively, these investigations show that a large proportion ofdiarrheal diseases in children and also in adults are associated withpathogenic E. coli that are falsely diagnosed as commensal flora instandard microbiological procedures. The TAQMAN™ methodology accordingto the invention for the first time enables the direct, fast, specific,and sensitive detection of these important pathogens. Moreover,virulence genes detected with this approach are not confined to E. coli,they also can be freely transmitted to other enterobacteria. Detectionof the virulence genes within these bacteria would also be covered bythe herein described TAQMAN™-PCR The assay requires only minimalpost-PCR detection time, can thus be performed under 18 hours, andabolishes PCR-cross contamination problems.

According to the present invention E. coli virulence factor/toxin geneswere used as targets for PCR amplification. PCR primers and fluorogenicprobes were designed on the basis of published sequences. Eightdifferent primer and probe sets for detection of pathogenic groups of E.coli and related enterobacteria were specifically chosen, see table 1.

Primer sequences and their locations with GenBank accessions aredetailed in Table 2. Detection of EHEC sltI is based on consensus primerand probe sequences after alignment of sltI homologous genes (Genbankaccessions Z36899, Z36900, and Z36901) (77,78). Detection of sltIIvariants is based on published sequences of homologous genes (Genbankaccessions M76738, Z37725, L11079, X67515, M59432, M29153, M36727, andM21534) (79-83). For amplification of sltII, degenerate primer setsproved optimal. Diagnosis of ETEC is based on amplification of eitherheat labile (LT) (84) or heat stable toxin (ST) (36), EaggEC on pCVD432plasmid sequences (40,50), EIEC on inv-plasmid sequences (38,48), EPECon E. coli attaching and effacing gene (EAF plasmid) (37,85) or E. coligene for EHEC attaching and effacing protein (eae) (86). PCR controlamplification for integrity of DNA preparations was performed usingprimers specific for the E. coli parC gene (topoisomerase IV, Genbankaccession M58408) (87).

Oligonucleotide probes and their Genbank Ref. are shown in table 3.Oligonucleotide probes were designed (if possible) with a GC-content of40-60%, no G-nucleotide at the 5′-end, length of probes was 27 to 30 bp.Probes were covalently conjugated with a fluorescent reporter dye (e.g.6-carboxy-fluorescein [FAM]; λ_(em)=518 nm) and a fluorescent quencherdye (6-carboxytetram-ethyl-rhodamine [TAMRA]; λ_(em)=582 nm) at the most5′ and most 3′ base, respectively. All primers and probes were obtainedfrom Perkin Elmer, Germany.

TAQMAN™-PCR was optimized by isolation of DNA from E. coli controlstrains harboring genes for LT, ST, inv-plasmid, pCVD342, EAF, eae, sltIand sltII (see Table 1). MgCl₂ concentrations were adjusted for maximumPCR. product yields (as verified by agarose gel electrophoresis) and RQvalues (RQ=FAM_(fluorescence intensity)/TAMRA_(fluorescence intensity))with the above mentioned pathogenic E. coli control strains. Optimum PCRreactions for all primer/fluorigenic probes used were obtained at aMgCl₂ concentration of 5.2 mmol, 35 PCR cycles, an annealing temperatureof 55° C. and an extension temperature of 65° C. Extension at 65° C. wasfound to yield higher RQ values, probably due to a lower rate oftemplate/fluorogenic probe dissociation before degradation byTaq-polymerase.

The E. coli sltI gene was used as a target sequence for establishment ofPCR and analysing different locations of probes relative to the PCRprimers. Primers were designed to anneal in conserved regions of thesltI genes (see above). Two probes, sltI-N0 located 132 bp upstream ofone primer and sltI-N1, placed at a 21 bp distance from the primer werecompared. RQ values achieved with probe sltI-N1(RQ_(m)=6.3800) werereproducably found higher than RQ values generated with probe sltI-NO(RQ_(m)=0.9620) at equal template concentrations of the E. coli sltIcontrol DNA. Generally, also probes specific for other target genes thatwere located close (4 to 20 bp) to one of the two PCR primers yieldedconsistently higher RQ values than probes that were placed at a greaterdistance from the primers.

The influence of DNA preparation on the performance of TAQMAN™-PCR wastested, since it has been reported that crude bacterial lysates cancontain inhibiting factors that might interfere with PCR performance.Therefore, bacteria were collected after overnight growth on McConkeyplates. DNA was prepared by boiling of bacteria inoculated in 0.9% NaClsolution or by isolation of genomic DNA with a commercial spin prepprocedure (see the example, material and methods). The RQ values andsensititvity of TAQMAN™-PCR did not differ when the two preparationmethods were compared. The RQ values obtained for PCR amplificationsfrom DNA derived from 10⁵ Sltl or sltII containing EHEC prepared byboiling or by spin prep comparable.

The TAQMAN™-PCR method relies on the detection of free reporter dye(FAM) that is released from the probe after hydrolysis. Thus, probeconcentration should also have an effect on the assay performance byaffecting the fraction of the probe that is degraded during PCR cycling.Probe concentrations were titrated in the range of 100 pmol to 0.1 pmoland ΔRQ values were determined. Optimal probe concentrations varied inbetween 10 pmol and 20 pmol depending on the target gene that wasamplified.

For testing sensitivity of TAQMAN™-PCR, EHEC containing either sltl orsltll were diluted in a suspension containing E. coli strain ATCC11775at 10⁷ CfU at log step dilutions. PCR was performed under optimizedconditions and results from ethidium-bromide stained agarose gels werecompared to TaqMan™ results. Minimum detection limits of a sltlcontaining EHEC strain was 10³ cfu within 10⁷. For sltII the detectionlimit was found at 10^(3.5) cfu in 10⁷ enterobacteria. Both methods,detection of PCR products by agarose gel electrophoresis and measurementof fluorescence signals by the TaqMan method yielded comparable results,i.e. that at ΔRQ values above ΔRQ_(threshold) PCR product bands werevisible in agarose gelb whereas at ΔRQ values around ΔRQ threshold alsoin agarose gels PCR products were below the detection limit. Afteroptimizing detection tests for all virulence factors/toxins, TAQMAN™-PCRwas set up for routine testing of biological specimen for the presenceof pathogenic E. coli bacteria. Results of TAQMAN™-PCR were compared toagarose gel electrophoresis.

The following example will illustrate the invention further. It is,however, not to be construed as limiting.

EXAMPLE

1. Prevalence of Pathogenic E. coli in Stool Specimens from Childrenwith Diarrhea was Tested Using the Method According to the Invention.

In order to verify TAQMAN™-PCR performance and to test for the occurenceof pathogenic E. coli screening of 100 stool specimens from children ofage 0 to 10 years with the clinical symptoms of diarrhea was undertaken.The materials and methods used in the test are described in more detailbelow under item 2.

Collection of specimen took place from June to October 1996. All samplesin this study were derived from the area of Southern Bavaria. Stoolspecimen were plated on McConkey agar, incubated overnight andenterobacteria were collected. DNA was isolated and used as template inPCR reactions containing specific primers and fluorigenic probes forsltI, sltII, LT, ST, EAF-plasmid, eae-gene, inv-plasmid, and pCVD432.For verification of the integrity of DNA from individual preparations acontrol PCR reaction was set up, containing primers and an internalfluorigenic probe for amplification of the parC gene of E. coli. As apositive assay control, one PCR reaction was performed within eachassay, where DNA from a positive control strain for the respectivevirulence factor/toxin was present. Applying this method reliable,specific and sensitive detection of all target genes could be achieved.Systematic analysis of 100 stool specimen derived from childrensuffering from diarrhea yielded 22 samples where one, two or three ofthe virulence factors/toxins of pathogenic E. coli could be detected. Indetail, 2 patients harbored EHEC (one with hemorrhagic colitis and onedeveloped HUS). 3 patients tested positive for ETEC, 16 for EPEC, 1 forEIEC, and 8 for EaggEC (see Table 4). The patient suffering fromhemorrhagic colitis tested positive for sltI and eae, the patientdeveloping HUS tested positive for sltl, sltII and eae. One patientsimultaneously harbored ETEC (LT+,ST+), EPEC (eae+), and EaggEC(pCVD342+), one patient tested positive for EIEC (inv+) and EaggEC(pCVD342+), two stool specimen contained EPEC (eae+) and EaggEC(pCVD342).

Enterobacteria from the two patients with EHEC were hybridized with sltland sltII gene probes for testing accuracy and specificity ofTAQMAN™-PCR. In the case of patient one, where TAQMAN™-PCR was positivefor sltl, only colonies hybridizing with sltI could be found. Coloniesof patient two, where TAQMAN™-PCR was positive for sItI and sItII,hybridized with probes for sltI and sltII. Positive colonies were pickedand biochemically typed as E. coli.

Antibiotic susceptibilty testing revealed that EHEC strains weresensitive to broad spectrum penicillins, cephalosporins and gyraseinhibitors.

2. Materials and Methods

a) Bacterial strains, media, culture and DNA preparation: A number ofEHEC, ETEC, EPEC, EIEC, and EaggEC E. coli strains were used as controlsfor accurate PCR amplification and were kindly provided by H. Karch,Würzburg, Germany and H. Beutin, Berlin, Germany (see Table 1) As astrain not harboring these virulence genes E. coli ATCC 11775 was used.For TaqMan™-PCR optimization, positive control strains were grown onMcConkey agar (Becton Dickinson, Germany) at 37° C. After overnightculture, bacteria were collected and resuspended in 0.9% NaCl solution.Turbidity was adjusted to McFarland 0.5. DNA was either prepared byboiling (950° C, 10 min) or isolated using QiaAmp tissue kit spin prepcolumns (Qiagen, Germany). 10 μl of DNA suspension was used for PCR.Detection of pathogenic E. coli strains from stool specimen of humans orcows was performed after spreading an appropriate amount of stool onMcConkey plates. After overnight culture all bacterial colonies from thesurface of the McConkey plates were collected and processed as detailedabove.

b) PCR-cyling: PCR recations were set up in 70 μl final volume inthin-walled 0.2 ml “optical PCR-tubes” (Perkin Elmer, Germany). Thereaction mix contained: 10 μl of bacterial lysate, 5.25 μl 25 mmolMgCl₂, 7 μl 10×PCR buffer, 40 pmol primers, 20 pmol specific fluorogenicprobe, 150 μM of each dATP, dTTP, dGTP, dCTP (Perkin Elmer), 1 UAmpliTaq-Polymerase (Perkin Elmer). A Perkin Elmer model 9600 thermalcycler was used for PCR cycling. Initial denaturation of bacterial DNAwas performed by heating for 5 min to 94° C. All cycles included adenaturation step for 15 sec at 94° C., annealing for 1 min 30 sec at55° C., and extension for 1 min 30 sec at 65° C. 35 cycles wereperformed.

c) Post-PCR processing: After completion of cycling, the fluorescenceintensities of the reporter dye, FAM, and the quencher dye, TAMRA, weredetermined using a Perkin Elmer LS50B luminiscence spectrophotometerequipped with a plate reader and modified for fluorescence measurementsof PCR reactions in optical tubes. ΔRQ values were calculated asdescribed in (74). A ARQ_(threshold) value was calculated on the basisof a 99% confidence interval above the mean of the triplicate notemplate controls(ΔRQ_(threshold)=6,95×std_(mean of no template controls)). PCR reactionswere scored positive if ΔRQ_(sample)>ΔRQ_(threshold) was given. Forverification of the sensitivity of TaqMan™-measurements, PCR productswere subjected to agarose gel electrophoresis. 15 μl of sample wereloaded with 2 μl sample buffer. PCR products were separated in 2%agarose gels containing ethidium bromide at 100V for 35 min. DNA wasvisualized under UV light and a digital image file was obtained usingthe Eagle EyeII System (Stratagene).

d) Verification of PCR amplificates: PCR products obtained fromtemplates of respective positive control strains were directly subclonedinto the TA cloning vector (Invitrogen, Germany) for verification ofspecificity of PCR amplification. After transfection (CaCl₂-method) ofDH5α bacteria with the ligation products, plasmid containing bacteriawere selected on ampicillin (Sigma, Germany) containing LB plates.Plasmid DNA was purified with Qiagen DNA purification columns (Quiagen,Germany). Inserts were PCR-cycle sequenced employing dideoxy-nucleotidesconjugated to 4 dyes (DNA Dye terminator cycle sequencing kit, PerkinElmer, Germany). Sequences were obtained with an Applied Biosystemsmodel 373A (Applied Biosystems, Germany). Insert sequences were alignedto published sequences as referenced in Table 1 using the McDNAsisprogramme (Appligene, Great Britain). Sequence comparisons verified thatthe PCR products were identical to the respective virulence factors ortoxins.

e) Sensitivity of TAQMAN™ technique: For determination of thesensitivity of the TAQMAN™ method, serial log-step dilutions of positivecontrol strains were performed in a solution containing 10⁷ cfu of E.coli reference strain ATCC 11775 DNA was either prepared by the boilingmethod (see above) or purified using spin prep columns designed forisolation of genomic bacterial DNA (Qiagen, Germany). Purification wasaccording to the protocol of the manufacturer. The detection limit forsItI containing strains was determined with 10³ cfu among 10⁷ E. coliand for sItII containing strains as 10^(3.5) among 10⁷.

f) Colony hybridisation and isolation of EHEC bacteria: EHEC bacterialstrains and stool samples from patients testing positive in sltI orsItII TAQMAN™-PCR were subjected to colony hybridisation. Briefly,bacteria were plated on-McConkey agar plates such that single coloniescould be seen. Bacteria were blotted on nylon membranes (GenescreenPlus, NEN, Germany), cracked (1% SDS), denatured (0.5M NaOH, 1.5M NaCI),neutralized (1M TRIS, 1.5M NaCI), and washed (20×SSC). Membranes werebaked at 80° C. for 2 hours. DNA probes specific for sal or sltII werelabelled with fluorescein (Gene-Images random prime labelling module,Amersham, Germany). Afterwards, filters were hybridized with labelledprobes. Hybridization was verified by non-radioactive detection systememploying anti-FITC peroxidase mAb and ECL detection module (Gene ImagesCDP-Star detection module, Amersham, Germany). Bacterial colonieshybridizing with the probe and non-hybridizing colonies were picked,verified by TAQMAN™-PCR and tested for antibiotic susceptibility.Antibiotic susceptibility testing. EHEC and non-EHEC E. coli were pickedfrom McConkey plates after testing for sltl or sltII or both toxin genesin colony hybridazation and MIC testing was performed according to NCCLSguidelines for enterobacteria.

TABLE 1 E. coli strains - virulence factors/toxins Strain Group numberSerotype Virulence factor/toxin EHEC 1193/89 O157:H- sltI, ene 3574/92O157:H7 sltII, ene A9167C O157:H7 sltI, sltIIc, ene 5769/87 O157:H7sltI, sltII, eae 427/89 O157:H- sltI, sltIIc, ene 1249/87 O157:H7 sltII,sltIIc, ene ETEC 147/1 O128:H- ST 164/82 O148:H28 LT EPEC 111/87 O111EAF, ene 12810 O114:H2 EAF, eae EIEC 76-5 O143 inv-plasmid 12860 O124inv-plasmid EaggEC pCVD432 plasmid control ATCC 11775 —

TABLE 2 Primers for detection of pathogenic E. coli. W is A/T, R is A/G,D is A/G/T, Y is C/T and K is G/T. Virulence factor/ location Size ofGroup toxin Primer Sequence (5′ → 3′) of primer PCR product Genbank Ref.Ref. ETEC LT LT-1 gcg tta cta tcc tct 874-895 339 S60731 (84) cta tgt g(SEQ ID NO: 1) LT-2 agt ttt cca tac tga 1213-1192 ttg ccg c (SEQ ID NO:2) ST ST-1 tcc ctc agg atg cta 100-120 260 M34916 (36) aac cag (SEQ IDNO: 3) ST-2a tcg att tat tca aca 360-339 aag caa c (SEQ ID NO: 4) EaggECpCVD432 EA-1 ctg gcg aaa gac 66-87 629 X81423 (40, 50) plasmid tgt atcatt g (SEQ ID NO: 7) EA-2 taa tgt ata gaa atc 695-674 cgc tgt t (SEQ IDNO: 8) EIEC inv-plasmid EI-1 ttt ctg gat ggt atg 17786-17806 303 D50601(38, 48) gtg agg (SEQ ID NO: 9) emb EI-2 ctt gaa cat aag 18089-18069 gaaata aac (SEQ ID NO: 10) EPEC EAF plasmid EP-1 cag ggt aaa aga 546-568398 X76137 (37, 85) aag atg ata ag (SEQ ID NO: 11) EP-2 aat atg ggg acc944-923 atg tat tat c (SEQ ID NO: 12) eae EPeh-1 ccc gga ccc ggc  91-113872 Z11541 (86) aca agc ata ag (SEQ ID NO: 13) EPeh-2 agt ctc gcc agtatt 963-942 cgc cac c (SEQ ID NO: 14) EHEC sltI sltI-1 atg aaa aaa aca1113-1135 287 Z36899 (77, 78) tta tta ata gc (SEQ ID NO: 15) sltI-2 tcacyg agc tat tct 1400-1376 gag tca acg (SEQ ID NO: 16) sltII sltII-1 atgaag aag atr 1148-1178 265 L11079 (79-83) wtt rtd gcr sltII-2 gyt tta ttyg (SEQ ID NO: 17) 1413-1385 tca gtc atw att aaa ctk cac yts rgc aaa kcc(SEQ ID NO: 18) control parC par-1 aac ctg ttc agc gcc 141-161 260M58408 (87) gca ttg (SEQ ID NO: 28) par-2 aca acc ggg att 401-381 ccgtgt aac (SEQ ID NO: 29)

TABLE 3 TaqMan ™-probes used for detection of pathogenic E. colivirulence Gen- factor/ Probe for Taqman ™ bank Group toxin (FAM-5′ →3′-TAMRA) bp Ref. Ref. ETEC LT agc tcc cca gtc tat tac aga act atg 903-S60731 (84) (SEQ ID NO:19) 929 ST aca tac gtt aca gac ata atc aga atc ag334- M34916 (36) (SEQ ID NO:20) 306 EaggEC pCVD432 ctc ttt taa ctt atgata tgt aat gtc tgg 668- X81423 (40, 50) plasmid (SEQ ID NO:22) 639 EIECinv - caa aaa cag aag aac cta tgt cta cct 18063- D50601 (38, 48) plasmid(SEQ ID NO:23) 18037 emb EPEC EAF - ctt gga gtg atc gaa cgg gat cca aat575- X76137 (37, 85) plasmid (SEQ ID NO:24) 601 eae taa acg ggt att atcacc aga aaa atc c 935- Z11541 (86) (SEQ ID NO:25) 908 EHEC sltI tcg ctgaat ccc cct cca tta tga cag gca 1367- Z36899 (77, 78) (SEQ ID NO:26)1338 sltII cag gta ctg gat ttg att gtg aca gtc att 1371- L11079 (79-83)(SEQ ID NO:27) 1342 control parC atg tct gaa ctg ggc ctg aat gcc agc169- M58408 (87) (SEQ ID NO:30) 199

TABLE 4 Frequency of pathogenic E. coli in stool samples of childrenwith diarrhea (n = 100) Agar gel TaqMan: electrophores virulence numberof is: number of factor/ positive positive Group toxin isolates isolatespathogenic group ETEC LT 2 2 5 ST 3 3 EaggEC 60 kb 8 8 8 plasmid EIECinv plasmid 1 1 1 EPEC EAF plasmid 1 1 16 eae 15 15 EHEC sltI 2 2 2sltII 1 1 control parC 100 100

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30 1 22 DNA Escherichia coli CDS (1)...(22) 1 gcg tta cta tcc tct ctatgt g 22 Ala Leu Leu Ser Ser Leu Cys 1 5 2 22 DNA Escherichia coli CDS(1)...(22) 2 agt ttt cca tac tga ttg ccg c 22 Ser Phe Pro Tyr * Leu Pro1 5 3 21 DNA Escherichia coli CDS (1)...(21) 3 tcc ctc agg atg cta aaccag 21 Ser Leu Arg Met Leu Asn Gln 1 5 4 22 DNA Escherichia coli CDS(1)...(22) 4 tcg att tat tca aca aag caa c 22 Ser Ile Tyr Ser Thr LysGln 1 5 5 21 DNA Escherichia coli CDS (1)...(21) 5 aac tgc tgg gta tgtggc tgg 21 Asn Cys Trp Val Cys Gly Trp 1 5 6 21 DNA Escherichia coli CDS(1)...(21) 6 tgc tga cct gcc tct tcc atg 21 Cys * Pro Ala Ser Ser Met 15 7 22 DNA Escherichia coli CDS (1)...(22) 7 ctg gcg aaa gac tgt atc attg 22 Leu Ala Lys Asp Cys Ile Ile 1 5 8 22 DNA Escherichia coli CDS(1)...(22) 8 taa tgt ata gaa atc cgc tgt t 22 * Cys Ile Glu Ile Arg Cys1 5 9 21 DNA Escherichia coli CDS (1)...(21) 9 ttt ctg gat ggt atg gtgagg 21 Phe Leu Asp Gly Met Val Arg 1 5 10 21 DNA Escherichia coli CDS(1)...(21) 10 ctt gaa cat aag gaa ata aac 21 Leu Glu His Lys Glu Ile Asn1 5 11 23 DNA Escherichia coli CDS (1)...(23) 11 cag ggt aaa aga aag atgata ag 23 Gln Gly Lys Arg Lys Met Ile 1 5 12 22 DNA Escherichia coli CDS(1)...(22) 12 aat atg ggg acc atg tat tat c 22 Asn Met Gly Thr Met TyrTyr 1 5 13 23 DNA Escherichia coli CDS (1)...(23) 13 ccc gca ccc ggc acaagc ata ag 23 Pro Ala Pro Gly Thr Ser Ile 1 5 14 22 DNA Escherichia coliCDS (1)...(22) 14 agt ctc gcc agt att cgc cac c 22 Ser Leu Ala Ser IleArg His 1 5 15 23 DNA Escherichia coli CDS (1)...(23) 15 atg aaa aaa acatta tta ata gc 23 Met Lys Lys Thr Leu Leu Ile 1 5 16 24 DNA Escherichiacoli CDS (1)...(24) 16 tca cyg agc tat tct gag tca agc 24 Ser Xaa SerTyr Ser Glu Ser Ser 1 5 17 31 DNA Escherichia coli CDS (1)...(31) 17 atgaag aag atr wtt rtd gcr gyt tta tty g 31 Met Lys Lys Xaa Xaa Xaa Xaa XaaLeu Phe 1 5 10 18 33 DNA Escherichia coli CDS (1)...(33) 18 tca gtc atwatt aaa ctk cac yts rgc aaa kcc 33 Ser Val Xaa Ile Lys Xaa His Xaa XaaLys Xaa 1 5 10 19 27 DNA Escherichia coli CDS (1)...(27) 19 agc tcc ccagtc tat tac aga act atg 27 Ser Ser Pro Val Tyr Tyr Arg Thr Met 1 5 20 29DNA Escherichia coli CDS (1)...(29) 20 aca tac gtt aca gac ata atc agaatc ag 29 Thr Tyr Val Thr Asp Ile Ile Arg Ile 1 5 21 30 DNA Escherichiacoli CDS (1)...(30) 21 atg aag ggg cga agt tct ggc tca atg tgc 30 MetLys Gly Arg Ser Ser Gly Ser Met Cys 1 5 10 22 30 DNA Escherichia coliCDS (1)...(30) 22 ctc ttt taa ctt atg ata tgt aat gtc tgg 30 Leu Phe *Leu Met Ile Cys Asn Val Trp 1 5 23 27 DNA Escherichia coli CDS(1)...(27) 23 caa aaa cag aag aac cta tgt cta cct 27 Gln Lys Gln Lys AsnLeu Cys Leu Pro 1 5 24 27 DNA Escherichia coli CDS (1)...(27) 24 ctt ggagtg atc gaa cgg gat cca aat 27 Leu Gly Val Ile Glu Arg Asp Pro Asn 1 525 28 DNA Escherichia coli CDS (1)...(28) 25 taa acg ggt att atc aac agaaaa atc c 28 * Thr Gly Ile Ile Asn Arg Lys Ile 1 5 26 30 DNA Escherichiacoli CDS (1)...(30) 26 tcg ctg aat ccc cct cca tta tga cag gca 30 SerLeu Asn Pro Pro Pro Leu * Gln Ala 1 5 27 30 DNA Escherichia coli CDS(1)...(30) 27 cag gta ctg gat ttg att gtg aca gtc att 30 Gln Val Leu AspLeu Ile Val Thr Val Ile 1 5 10 28 21 DNA Escherichia coli CDS (1)...(21)28 aac ctg ttc agc gcc gca ttg 21 Asn Leu Phe Ser Ala Ala Leu 1 5 29 21DNA Escherichia coli CDS (1)...(21) 29 aca acc ggg att cgg tgt aac 21Thr Thr Gly Ile Arg Cys Asn 1 5 30 30 DNA Escherichia coli CDS(1)...(30) 30 atg tct gaa ctg ggc ctg aat gcc agc gcc 30 Met Ser Glu LeuGly Leu Asn Ala Ser Ala 1 5 10

What is claimed is:
 1. A method for detection and differentiation ofpathogenic enterobacteria in a sample, said method comprising: isolatingnucleic acid from said sample; adding a set of oligonucleotide primerpairs to said nucleic acid sample wherein said set of oligonucleotideprimers comprises at least five oligonucleotide primer pairs wherein,wherein each primer pair specifically amplifies a DNA sequence of avirulence factor/toxin gene characteristic for one each of the subgroupsof the pathogenic E. coli strains, said subgroups comprisingenterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic andenterohemorrhagic E. coli strains and wherein for amplification of eachsubgroup at least one oligonucleotide primer pair is included in saidset of oligonucleotide primer pairs, and wherein the set comprises aprimer pair that hybridizes to an inv-plasmid of enteroinvasive E. coliand wherein said primer pair is El-1: 5″TTT CTG GAT GGT ATG GTG AGG 3′(SEQ ID NO: 9) and E1-2: 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO:10) which specifically amplifies a DNA sequence of an inv plasmidcharacteristic for enteroinvasive E. coli; and subjecting said nucleicacid and said set of primer pairs to an amplification process; anddetecting the presence of at least one amplified product, wherein thepresence of at least one amplified product indicates the presence of atleast one pathogenic enterobactcria strain in said sample.
 2. The methodaccording to claim 1, wherein the set of oligonucleotide primer pairscomprises primer pairs selected from the group consisting of: at leastone primer pair that hybridizes to a gene encoding heat labile toxin ora gene encoding heat stabile toxin for amplification of a DNA sequencecharacteristic for enterotoxigenic E. coli; at least one primer pairthat hybridizes to a gene encoding heat stabile toxin or to a pCDVD432plasmid for amplification of a DNA sequence characteristic forenteroaggregative E. coli; at least one primer pair that hybridizes to aEAF plasmid, or an eae gene for amplification of a DNA sequencecharacteristic for enteropathogenic E. coli; and at least one primerpair that hybridizes to genes encoding shiga-like toxin sltI or sltIIfor amplification of a DNA sequence characteristic for enterohemorrhagicE. coli.
 3. The method according to claim 2, wherein the oligonucleotideprimer pair that hybridizes to the gene encoding heat labile toxincharacteristic for enterotoxigenic E. coli is LT-I: 5′GCG TTA CTA TCCTCT CTA TGT G 3′ (SEQ ID NO: 1) and LT-2: 5′AGT TTT CCA TAC TGA TTG CCGC 3′ (SEQ ID NO: 2); and the oligonucleotide primer pair that hybridizesto the gene encoding heat stabile toxin characteristic forenterotoxigenic E. coli is ST-1: 5′TCC CTC AGG ATG CTA AAC CAG 3′ (SEQID NO: 3) and ST-2a:5′TCG ATT TAT TCA ACA AAG CAA C 3′ (SEQ ID NO: 4);and the oligonucleotide primer pair that hybridizes to the gene encodingheat stabile toxin characteristic for enteroaggregative E. coli isEASTI-1: 5′AAC TGC TGG GTA TGT GGC TGG 3′ (SEQ ID NO: 5) and EASTI-2:5′TGC TGA CCT GCC TCT TCC ATG 3′ (SEQ ID NO: 6); and the oligonucleotideprimer pair which hybridizes to the pCVD432 plasmid is EA-1: 5′CTG GCGAAA GAC TGT ATC ATT G3′ (SEQ ID NO: 7) and EA-2: 5′TAA TGT ATA GAA ATCCGC TGT T3′ (SEQ ID NO: 8); and the oligonucleotide primer pair whichhybridizes to the EAF plasmid is EP-1: 5′CAG GGT AAA AGA AAG ATG ATAAG3′ (SEQ. ID NO: 11) and EP-2:5′AAT ATG GGG ACC ATG TAT TAT C 3′ (SEQID NO: 12); and the oligonucleotide primer pair which hybridizes to theeae gene is EPeh-1: 5′CCC GCA CCC GGC ACA AGC ATA AG 3′ (SEQ ID NO: 13)and EPeh-2: 5′AGT CTC GCC AGT ATT CGC CAC C 3′ (SEQ ID NO: 14); and theoligonucleotide primer pair which hybridizes to the gene encodingshiga-like toxin SltI is SltI-1: 5′ATG AAA AAA ACA TTA TTA ATA GC 3′(SEQ ID NO: 15) and SltI-2: 5′TCA CYG AGC TAT TCT GAG TCA AGC 3′ (SEQ IDNO: 16); and the oligonucleotide primer pair which hybridizes to thegene encoding shiga-like toxin SltII is SltII-1: 5′ATG AAG AAG ATR WTTRTD GCR CYT TTA TTY G3′ (SEQ ID NO:17) and SltII-2: 5′TCA GTC ATW ATTAAA CTK CAC YTS RGC AAA KCC 3′ (SEQ ID NO: 18), wherein W is AT, R isA/G, D is A/G/T, Y is C/T and K is G/T.
 4. The method according toclaim, wherein detecting the presence of at least one amplified productis performed using at least one oligonucleotide probe capable ofhybridizing to the amplified product wherein said oligonucleotide probeis labeled at the 5′end with a fluorescent reporter dye and at the 3′end with a fluorescent quencher dye and is susceptible to 5′-3′exonuclease degradation by a polymerase, and wherein said amplificationprocess uses a polymerase having 5′-3′ exonuclease degradation activity.5. The method according to claim 4 wherein the labeled oligonucleotideprobe is selected from the group consisting of: a labeledoligonucleotide probe specific for the detection of a heat labile toxingene characteristic for enterotoxigenic E. Coli; a labeledoligonucleotide probe specific for the detection of a heat stabile toxingene characteristic for enterotoxigenic E. Coli; a labeledoligonucleotide probe specific for the detection of a heat stabile toxingene characteristic for enteroaggregative E. Coli; a labeledoligonucleotide probe specific for the detection of a pCVD432 plasmid; alabeled oligonucleotide probe specific for the detection of an invplasmid; a labeled oligonucleotide probe specific for the detection of aEAF-plasmid; a labeled oligonucleotide probe specific for the detectionof a eae gene; a labeled oligonucleotide probe specific for thedetection of a shiga-like toxin SltI gene; and a labeled oligonucleotideprobe specific for the detection of a shiga-like toxin SltII gene. 6.The method according to claim 5, wherein the labeled oligonucleotideprobe for the detection of heat labile toxin gene characteristic forenterotoxigenic E. coli is 5′AGC TCC CCA GTC TAT TAC AGA ACT ATG 3′ (SEQID NO: 19), the labeled oligonucleotide probe for the detection of heatstabile toxin gene characteristic for enterotoxigenic E. coli is 5′ACATAC GTT ACA GAC ATA ATC AGA ATC AG 3′ (SEQ ID NO: 20); the labeledoligonucleotide probe for the detection of heat stabile toxin genecharacteristic for enteroaggregative E. coli is 5′ATG AAG GGG CGA AGTTCT GGC TCA ATG TGC 3′ (SEQ ID NO: 21); the labeled oligonucleotideprobe for the detection of pCVD432 plasmid is 3′ CTC TTT TAA CTT ATG ATATGT AAT GTC TGG 3′ (SEQ ID NO: 22); the labeled oligonucleotide probefor the detection of the inv-plasmid is 5′CAA AAA CAG AAG AAC CTA TGTCTA CCT 3′ (SEQ ID NO: 23) the labeled oligonucleotide probe for thedetection of the EAF-plasmid is 5′CTT GGA GTG ATC GAA CGG GAT CCA AAT 3′(SEQ ID NO: 24); the labeled oligonucleotide probe for the detection ofthe eae gene is 5′TAA ACG GGT ATT ATC AAC AGA AAA ATC C 3′ (SEQ ID NO:25); the labeled oligonucleotide probe for the detection of shiga-liketoxin SltI gene is 5′TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA 3′ (SEQ IDNO: 26); and the labeled oligonucleotide probe for the detection ofshiga-like toxin SltII gene is 5′CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT3′ (SEQ ID NO: 27).
 7. The method according to claim 4, wherein thefluorescent reporter dye is 6-carboxy-fluorescein,tetrachloro-6-carboxy-fluorescein, or hexachloro-6-carboxy-fluorescein,and the fluorescent quencher dye is 6-carboxytetramethyl-rhodamine. 8.The method according to claim 1 wherein the amplification processcomprises 35 PCR cycles at a MgCl₂ concentration of 5.2 mM, an annealingtemperature of 55° C. and an extension temperature of 65° C.
 9. A set ofoligonucleotide primer pairs useful for polymerase chain reaction (PCR)amplification of pathogenic enterobacteria allowing detection anddifferentiation of pathogenic enterobacteria in a sample whereinfollowing amplification the presence of at least one amplified productindicates the presence of at least one pathogenic enterobacteria strainin said sample, wherein said set comprises at least five primer pairs,wherein each primer pair specifically amplifies a DNA sequence of avirulence factor/toxin gene characteristic for one each of the subgroupsof the pathogenic E. coli strains, said subgroups comprisingenterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic andenterohemorrhagic E. coli strains and wherein for amplification of eachsubgroup at least one oligonucleotide primer pair is included in saidset of oligonucleotide primer pairs, and wherein the set comprises aprimer pair that hybridizes to an inv-plasmid of enteroinvasive E. coliand wherein said primer pair is El-1: 5′TTT CTG GAT GGT ATG GTG AGG 3′(SEQ ID NO: 9) and E1-2: 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO:10).
 10. The set of primer pairs according to claim 9 comprising aprimer pair that hybridizes to a gene encoding heat labile toxin, or toa gene encoding heat stabile toxin of enterotoxigenic E. coli; a primerpair that hybridizes to a gene encoding heat stabile toxin or to apCVD432 plasmid of enteroaggregative E. coli; a primer pair thathybridizes to a EAF plasmid, or a eae gene of enteropathogenic E. coli;and a primer pair that hybridizes to a gene encoding shiga-like toxinsltl or sltII of enterohemorrhagic E. coli.
 11. The set of primer pairsaccording to claim 10 wherein the primer pair which hybridizes to thegene encoding heat labile toxin of enterotoxigenic E. coli is LT-1:5′GCG TTA CTA TCC TCT CTA TGT G 3′ (SEQ ID NO: 1) and LT-2: 5′AGT TTTCCA TAC TGA TTG CCG C 3′ (SEQ ID NO: 2); the primer pair whichhybridizes to the gene encoding heat stabile toxin of enterotoxigenic E.coli is ST-1: 5′TCC CTC AGG ATG CTA AAC CAG 3′ (SEQ ID NO: 3) and ST-2a:5′TCG ATT TAT TCA ACA AAG CAA C 3′ (SEQ ID NO: 4); the primer pair whichhybridizes to the gene encoding heat stabile toxin of enteroaggregativeE. coli is EASTI-1: 5′AAC TGC TGG GTA TGT GGC TGG 3′ (SEQ ID NO: 5) andEASTI-2: 5′TGC TGA CCT GCC TCT TCC ATG 3′ (SEQ ID NO: 6); the primerpair which hybridizes to the pCVD432 plasmid is EA-1: 5′CTG GCG AAA GACTGT ATC ATT G 3′ (SEQ ID NO: 7) and EA-2: 5′TAA TGT ATA GAA ATC CGC TGTT3′ (SEQ ID NO:8); the primer pair which hybridizes to the EAF plasmidis EP-1: 5′CAG GGT AAA AGA AAG ATG ATA AG 3′ (SEQ ID NO: 11) and EP-2:5′AAT ATG GGG ACC ATG TAT TAT C 3′ (SEQ ID NO: 12); the primer pairwhich hybridizes to the eae gene is EPeh-1: 5′CCC GGA CCC GGC ACA AGCATA AG 3′ (SEQ ID NO: 13) and EPeh-2: 5′AGT CTC GCC AGT ATT CGC CAC C 3′(SEQ ID NO: 14); the primer pair which hybridizes to the shiga-liketoxin sltI gene is SltI-1: 5′ATG AAA AAA ACA TTA TTA ATA GC 3′ (SEQ IDNO: 15) and SltI-2; 5′TCA CYG AGC TAT TCT GAG TCA AGC 3′ (SEQ ID NO:16); and the primer pair which hybridizes to the shiga-like toxin sltIIis SltII-1: 5′ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G 3′ (SEQ ID NO:17) and SltII-2: 5′TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC 3′ (SEQID NO: 18) wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K isG/T.
 12. A set of oligonucleotide primer pairs an a set ofoligonucleotide primer probes useful for diagnosing an enterobacteriainfection in samples derived from a living animal body including ahuman, by real time PCR method, wherein said sets of oligonucleotideprimer pairs and oligonucleotide probes allow detection anddifferentiation of pathogenic enterobacteria in a sample, wherein saidset of oligonucleotide primer pairs comprises at least five primerpairs, wherein each primer pair specifically amplifies a DNA sequence ofa virulence factor/toxin gene characteristic for one each of thesubgroups of the pathogenic E. coli strains, said subgroups comprisingenterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic andenterohemorrhagic E. coli strains and wherein for amplification of eachsubgroup at least one oligonucleotide primer pair is included in saidset of oligonucleotide primer pairs, and wherein the set comprises aprimer pair that hybridizes to an inv-plasmid of enteroinvasive E. coli,wherein said primer pair is El-1: 5ΔTTT CTG GAT GGT ATG GTG AGG 3′ (SEQID NO: 9) and E1-2: 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO: 10).13. The method of claim 1, wherein said method is used to diagnose anenterobacteria infection in a sample derived from a living animal body.14. The method of claim 13, wherein said sample is derived from a human.15. The method of claim 1, wherein said method is used to detectenterobacteria contamination of a consumable.
 16. The method of claim15, wherein said consumable is selected from the group consisting ofmeat, milk and vegetable.